(1) Field of the Invention
This invention relates generally to the genesis and malignancy of tumors, and more particularly to methods for screening substances for oncogenic activity and to methods for assessing tumor aggressiveness.
(2) Description of the Related Art
Almost a century ago, Paul Erlich suggested that the immune system played a role in the elimination of spontaneously arising tumor cells (Ned. Tiijdschr. Geneeskd. 5(Pt. 1): 273, 1909, incorporated herein by reference). In 1970, Burnet coined the term "immune surveillance" to embody this concept and proposed that T cells would function as the major effectors in this system (Progr. Exp. Tumor Res. 13:1, 1970, incorporated herein by reference).
Recently, insight into the nature of the immune responses to tumors that were not nascently formed was provided by a study which suggested that the cytokine interferon.gamma. (IFN.gamma.) plays an important role in promoting tumor cell recognition and elimination (Dighe et al., Immunity 1:447-456, 1994, which is incorporated herein by reference). In this study, tumor cells derived from two methylcholanthrene-induced murine sarcomas in genetically different mice were transfected with a plasmid encoding a cytoplasmically truncated form of the murine IFN.gamma.-receptor ligand binding chain (.alpha. chain). These cell lines are completely unresponsive to IFN.gamma. due to overexpression of the functionally inactive IFN.gamma.-receptor .alpha. chain at the cell surface. When these IFN.gamma.-insensitive tumor cells were inoculated subcutaneously at low amounts (1 to 2.times.10.sup.4 cells/mouse) into normal syngeneic mice, they formed rapidly progressing tumors in at least 80% of the injected mice. In contrast, mice inoculated with the same amount of IFN.gamma. sensitive wild type tumor cells did not develop tumors. Dighe et al concluded that the development of host responses to the tumor studied requires the production of IFN.gamma. by host cells, the capacity of the tumor to respond to IFN.gamma., and the development of specific T cell immunity.
Although this study identified a role for IFN.gamma. in promoting rejection of transplantable tumors, they did not address the critical question of whether IFN.gamma. participates in promoting host responses to nascently forming transformed cells, i.e., whether it is involved in promoting tumor surveillance. More importantly, the study did not address whether IFN.gamma. responsiveness of the host cell played a role in tumor surveillance, which would be of critical importance in the development of an oncogenic screening method.
In another study of the physiological role of IFN.gamma. in the immune response, knockout mice with an inactivated gene for the IFN.gamma. receptor .alpha. chain were made and shown to be viable with no apparent phenotypic anomalies by 12 months (Huang et al., Science 259:1742-1745, 1993, incorporated herein by reference). The immune system in these IFN.gamma.R.sup.-/- mice appeared to develop normally in that no differences in the major lymphocyte subpopulations between mutant and wild-type mice were observed. However, the mutant mice had increased susceptibility to infection by Listeria monocytogenes and vaccinia virus despite normal cytotoxic and T helper cell responses. In addition, while the IFN.gamma.-unresponsive mice generated a normal antigen-specific IgM and IgGl response, they failed to develop a normal IgG2a response as indicated by decreased titers of antigen-specific IgG2a antibodies at twelve days after immunization with antigen.
Increased susceptibility to infection by microbial pathogens and viruses was also observed in knockout mice deficient in STAT1, an IFN-specific cytosolic transcription factor in the JAK-STAT signaling pathway (Meraz, et al., Cell 84:431-442, 1996, incorporated herein by reference). While these STAT1-deficient mice showed no overt developmental abnormalities and had normal populations and subpopulations of T cells, B cells, and macrophages, they died after infection with doses of Listeria monocytogenes and VSV that are sublethal in normal mice. Cells derived from the mutant mice were not responsive to IFN.alpha. and IFN.gamma. but did respond normally to other cytokine ligands, including growth hormone, epidermal growth factor, and interleukin-10.
A recent study examined the susceptibility of perforin-deficient (PKO) mice to tumor induction by a sarcoma-inducing carcinogen, methylcholanthrene (MCA), a papilloma-inducing carcinogen, 7,12-dimethylbenzanthracene (DBMA) plus 12-O-tet-radecanoyl-phorbol-13-acetate (TPA) (DBMA+TPA), and a sarcoma-inducing virus, Moloney murine sarcoma virus (MOMSV) (van den Broek et al., J. Exp. Med., 184:1781-1790, 1996). PKO mice subcutaneously injected with MCA developed sarcomas at the injection site at a higher frequency and with accelerated onset of tumor than observed in normal MCA-treated mice. In addition, although PKO and normal mice injected intramuscularly with MOMSV displayed similar numbers and kinetics of tumor onset, MOMSV-induced tumors were larger and regression was retarded in the PKO mice than in normal mice. However, the incidence and kinetics of DMBA+TPA-induced papillomas were similar in PKO and normal mice. The authors concluded that several mechanisms probably control tumor growth, with perforin-mediated cytotoxicity playing a role in some types of tumors, but not in others.
Assessing the carcinogenic potential of chemical compounds is indispensable in drug development and in identifying environmental carcinogens. Currently, the gold standard carcinogenicity test is the rodent bioassay performed by the National Toxicology Program (NTP) at Research Triangle Park, N.C. (Ashby et al., Mutat. Res., 257: 229-306, 1991, incorporated herein by reference). The NTP rodent bioassay lasts more than two years, requires a large number of experimental animals, a large amount of laboratory space for animal testing, and a large number of laboratory technicians. The cost of the NTP bioassay is so high that only a few chemicals per year can be evaluated. However, there are many chemicals in commercial use or in the environment that have not been tested, and thousands of new chemicals are synthesized every year. Thus, there is much interest in developing improved animal bioassays that can evaluate the oncogenic potential of chemical compounds within a relatively short period of time.
Recently, in a brief reference to unpublished observations, Bach et al. (Annu. Rev. Immunol. 15:563-591, 1997, incorporated herein by reference) stated that the chemical carcinogen 3-Methylcolanthrene produced more tumors in a chain knockout mice than in wild-type controls. This reference discussed the role of IFN.gamma. in host surveillance. Nevertheless, the authors did not provide any suggestion as to whether the knockout mice or wild-type controls could be used in a carcinogenicity screening model nor did they provide sufficient details of their findings to allow one to assess the possibility of such use.
Genetically modified mice that carry specific oncogenes or inactivated tumor-suppressor genes have been proposed as candidate animal models for rapid carcinogenicity testing. (See, e.g., Tennant et al., Envir. Health Perspect. 103:942-950, 1995; Yamamoto et al., Carcinogenesis 17:2455-2461, 1996; and Berns, U.S. Pat. No. 5,174,986, each of which is incorporated herein by reference). Mutant mice that reportedly respond more rapidly and at higher frequency to various carcinogens than wild-type mice include v-Ha-ras transgenic mice (Tennant et al., supra), c-Ha-ras transgenic mice (Yamamoto et al., supra), transgenic mice that overexpress the pim-1 oncogene in lymphoid tissues (Storer et al., Carcinogenesis 16:285-293, 1995, incorporated herein by reference and Berns, supra), and p53 heterozygous knockout mice (Tennant et al., supra).
These known animal models have characteristics that may limit their use in a carcinogenic screening assay. For example, the p53.sup.-/+ mice apparently respond differently to different classes of carcinogens in that tumors were induced by the mutagenic carcinogens p-cresidine and 4-vinyl-1-cyclohexene diepoxide (VCD), but not by the nonmutagenic carcinogens N-methylolacrylamide (NMOA) and reserpine (Tennant et al., supra). In addition, transgenic mice that express an oncogene in only one tissue may not be able to detect the broadest range of tissue specific carcinogenic activity. Indeed, it was recently suggested that data showing a very weak lymphoma response by pim-1 mice to three genotoxic carcinogens that do not normally induce lymphomas and a negative response to a known mouse lymphomagen raised concern that pim-1 mice are not sufficiently sensitive to established carcinogens to justify their routine use in a short-term carcinogenic screening assay (Storer et al, supra). Finally, data obtained with transgenic mice having inappropriate expression of an oncogene may not be predictive of the effect of such compounds in animals with normal expression of the oncogene. Therefore, it would be desirable to develop a screening assay that uses an animal model in which tumors are rapidly induced by a wide variety of mutagenic and nonmutagenic carcinogens that act on different target tissues and through different mechanisms.